Introduction: Sexuality education is one of the most important educational needs for adolescents, and mothers may have the key role in educating these issues to their daughters. This study aimed at developing, implementing and evaluating of a training program for mothers to educate sexuality matters to their adolescent girls in Mahshahr, Iran. Methods: This field trial was conducted on 140 mothers having 12 to18-year girls from September to March 2012. Data was collected by two validated questionnaires. The first one assessed the “Parent-Child Relationship: Mother" questionnaire which indirectly measures the level of communication skills for mothers completed by the girls, and the second one measured "Knowledge and attitude about sexuality education to adolescents" questionnaire completed for mothers. Based on the results, an educational intervention for mothers consisted of three training sessions was designed and established. Before and three-month later data were analyzed and compared. Results: Three months after the intervention, the mean scores of mothers’ knowledge and attitude scores toward sexuality matters, was increased significantly (P <0.01). However, there was not any significant difference between the scores of mothers’ communication skills (P = 0.37).Conclusion: It seems that the designed educational program was effective to improve the knowledge and attitudes toward the sexuality matters in mothers, but learning communication skills as an important factor in training women for sexuality education to their adolescent girls, need to continuous, regular, and companion with experiences and practice.